Camptothecin (CPT) and its clinical analogs such as topotecan (TPT) and CPT-11 are a new class of chemotherapeutic agents with a novel mechanism of action targeting the nuclear enzyme topoisomerase I (topoisomerase), causing single and double strand-DNA breaks and subsequent cell death (for reviews, see Dancey J. et al. "Current perspectives on camptothecins in cancer treatment." Br. J. Cancer 74:327-338 (1996) and Sinha B. K., "Topoisomerase I inhibitors: A review of their therapeutic potential in cancer," Drugs 49:11-16 (1995)). The cytotoxicity of these agents is predominantly exerted during the S phase of the cell cycle (Darzynkiewicz Z. et al., "The cell cycle effects of camptothecins." In: Pantazis P. et al. (eds)., The camptothecins from discovery to the patient, The New York Academy of Sciences, New York, vol. 803, pp. 93-101 (1996)). This inhibition is the result of a passive collision of the advancing DNA replication forks with the CPT-topoisomerase-DNA cleavable complexes which are expected to cause an arrest of DNA replication and to kill cells by generating DNA strand breaks (D'Apra P. et al., "Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons." Cancer Res. 50:6919-6924 (1990) and Ryan A. J. et al., "Camptothecin cytotoxicity in mammalian cells is associated with the interaction of persistent double strand breaks in replicating DNA," Nucleic Acid Res. 19:3295-3300 (1991)).
The sensitivity of cells to CPT and its analogs can not be completely explained by the collision model. Recent evidence has indicated that the sensitivity of cells to CPT is also determined by their ability to activate checkpoints in the S and G.sub.2 phases of the cell cycle (Jones C. B. et al., "Sensitivity to camptothecin of human breast cancer cells and normal bovine endothelial cells, in vitro," Cancer Chemother. Pharmacol. (in press, 1997); O'Connor P. M. et al., "S-phase population analysis does not correlate with the cytotoxicity of camptothecin and 10,11-methyldioxycamptothecin in human colon carcinoma HT-29 cells," Cancer Commun. 3:233-240 (1991); Dubrez L. et al., "The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemia cells to topoisomerase I and II inhibitors," Leukemia 9:1013-1024 (1995); Wang Y. et al., "Down-regulation of DNA replication in extracts of camptothecin-treated cells: Activation of an S-phase checkpoint," Cancer Res. 57:1654-1659 (1997); and Goldwasser F. et al., "Correlations between S and G.sub.2 arrest and the cytotoxicity of camptothecin in human colon carcinoma cells," Cancer Res. 56:4430-4437 (1996)). This activation in S phase occurs with high doses of CPT and G.sub.2 /M when low doses of CPT are used, presumably to avoid high and low levels of DNA damage, respectively. DNA damage extends the time of at least two stages or checkpoints in the cell cycle, the G.sub.1 -S and the G.sub.2 -M transitions (Hartwell L. H. et al., "Checkpoints: controls that ensure the order of the cell cycle events," Science 246:629-634 (1989)). A critical component of the G.sub.1 checkpoint is the p53 gene product, which when inactivated by mutations, renders a cell incapable of G.sub.1 arrest following DNA damage (Kastan M. B. et al., "Participation of p53 protein in the cellular response to DNA damage," Cancer Res. 51:6304-6311 (1991); and Kuerbitz S. J. et al., "Wild-type p53 is a cell cycle checkpoint determinant following irradiation," Proc. Natl. Acad. Sci. USA 89:7491-7495 (1992)). Instead, cells arrest in G.sub.2 phase, (Barlogie B. et al., "Cell cycle stage-dependent induction of G.sub.2 phase arrest by different antitumor agents." Eur. J. Cancer 14:741-745 (1978); Soreson C. M. et al., "Influence of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G.sub.2 arrest and DNA double-strand breaks," Cancer Res. 48:4484-4488 (1988)). The G.sub.2 arrest can permit repair of DNA and ensures that DNA replication is complete before the cell enters into mitosis. Based on such findings it has been proposed and it is now largely accepted that the main function of normal p53 is to preserve genomic integrity by acting as the "guardian of the genome" (Lane, D. P., "p53, guardian of the genome," Nature (Lond.) 358:15-16 (1992)). As a consequence, tumor cells with no or mutated p53 function lose their sensitivity to a wide variety of DNA-damaging agent (Fan S. et al., "p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents." Cancer Res. 54:5824-5830 (1994)); Lee J. M. et al., "p53 mutations increase resistance to ionizing radiation," Proc. Natl. Acad. Sci. USA 90:5742-5746 (1993); and Lowe S. W. et al., "p53 status and the efficacy of cancer therapy in vivo." Science (Washington D.C.) 266:807-810 (1994)). It is possible that this observation occurs because p53 stimulates a more efficient DNA repair process. Therefore, treatment of tumor cells deficient in p53 normal function with topoisomerase inhibitors alone is unlikely to be curative, since G.sub.2 arrest induced by the use of low doses would allow DNA repair to occur prior to mitosis, thus preventing potentially lethal lesions from killing tumor cells, while S phase arrest induced with the use of high doses may inflict high levels of DNA damage on the normal cells. One way to increase the sensitivity of these tumor cells to DNA damaging agents is to modulate events at checkpoints in the S and G.sub.2 phases to which only damaged tumor cells with mutant p53 would progress. At the same time, normal cells that pass the G.sub.1 checkpoint during this modulation would also progress to G.sub.2 and would also be sensitive to modulation at S and G.sub.2 checkpoints. However, the wild-type p53 seems to protect these cells from abrogation at these checkpoints (Russell, K. J. et al., "Abrogation of the G.sub.2 checkpoint results in differential radiosensitization of G.sub.1 checkpoint-deficient and competent cells," Cancer Res. 55:1639-1642 (1995); and Powell, S. N. et al., "Differential sensitivity of p53- and p53+ cells to caffeine-induced radiosensitization and override of G.sub.2 delay," Cancer Res. 55:1643-1648 (1995)).
A variety of agents such as caffeine and other methylxanthines can override the DNA damage-dependent G.sub.2 -checkpoint and enhance drug-induced cytotoxicity (Russell K. J. et al., "Abrogation of the G.sub.2 checkpoint results in differential radiosensitization of G.sub.1 checkpoint-deficient and competent cells," Cancer Res. 55:1639-1642 (1995); and Fan S. et al., "Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline," Cancer Res. 55:1649-1654 (1995)). However, plasma drug concentrations higher than the maximum tolerated doses are required to achieve this effect in clinical settings. In search of new G.sub.2 -checkpoint inhibitors, a staurosporine analog, 7-hydroxystaurosporine (UCN-01), has been found to abrogate the cisplatin-induced S and G.sub.2 checkpoints and to enhance its cytotoxicity in CHO and HT-29 cells lacking normal p53 function (Bunch R. T. et al., "Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G.sub.2 -checkpoint inhibitor," Clinical Cancer Res. 2:791-797 (1996); and Wang Q. et al., "UCN-01: a potent abrogator of G.sub.2 checkpoint function in cancer cells with disrupted p53." JNCI 88:956-965 (1996)).